Ampure xp bead based clean up

T-cell receptor analysis was performed as described previously (54 (link)), with minor modifications. Briefly, mRNA was isolated with the RNA microkit (Qiagen) according to manufacturer’s protocol. Isolated mRNA was used for cDNA synthesis with 5’RACE template switch technology to introduce a universal primer binding site, and unique molecular identifiers (UMI’s) were added at the 5’ end of the cDNA molecules using the SMARTScribe Reverse Transcriptase (TaKaRa). cDNA synthesis was followed by an AMPure XP bead-based cleanup (Beckman Coulter). Purified cDNA molecules were amplified in two subsequent PCR steps using the Q5^® High-Fidelity DNA Polymerase (New England BioLabs), with an AMPure XP bead-based cleanup in between. PCR products were size selected on gel and purified using the Nucleospin PCR cleanup kit (Machery-Nagel). The PCR products were sequenced via Illumina MiSeq paired end using 2x250 bp sequencing.

T-cell receptor analysis was performed as described previously [23 (link)], with minor modifications. Briefly, mRNA was isolated with the RNA microkit (Qiagen) according to the manufacturer’s protocol. Isolated mRNA was used for cDNA synthesis with 5′RACE template switch technology to introduce universal primer binding sites, and unique molecular identifiers (UMI’s) were added at the 5′ end of the cDNA molecules using the SMARTScribe reverse transcriptase (TaKaRa). cDNA synthesis was followed by an AMPure XP bead-based clean-up (Beckman Coulter). Purified cDNA molecules were amplified in two subsequent PCR steps using the Q5^® High-Fidelity DNA Polymerase (New England BioLabs), with an AMPure XP bead-based clean-up step in between. PCR products were size-selected on gel and purified using the Nucleospin PCR clean-up kit (Machery-Nagel). The PCR products were sequenced via Illumina MiSeq paired-end 2 × 250 nt sequencing.

TCRβ analysis was performed as described previously (Shugay et al., 2014 (link)), with minor modifications. Briefly, mRNA was isolated with the RNA microkit (Qiagen) according to the manufacturer's protocol. Isolated mRNA was used for cDNA synthesis with 5′RACE template switch technology to introduce a universal primer binding site, and unique molecular identifiers (UMIs) were added at the 5′ end of the cDNA molecules using the SMARTScribe Reverse Transcriptase (TaKaRa). cDNA synthesis was followed by an AMPure XP bead-based clean-up (Beckman Coulter). Purified cDNA molecules were amplified in two subsequent PCR steps (25 cycles in PCR1 and 20 cycles in PCR2) using the Q5® High-Fidelity DNA Polymerase (New England BioLabs), with an AMPure XP bead-based clean-up in between. PCR products were size-selected on gel and purified using the Nucleospin PCR clean-up kit (Machery-Nagel). The PCR products were sequenced via Illumina MiSeq paired end 2x250 nucleotide (nt) sequencing.